Interpretive Summary: The yellow green molds, namely Aspergillus flavus and Aspergillus parasiticus, produce aflatoxin B1, which is one of the most potent cancer causing chemicals found in nature. When these fungi invade corn or cotton plants and produce aflatoxin, the crops become unmarketable. Our efforts have been focused on understanding the genetic and biochemical steps involved in the toxin synthesis. We hope to find simple non-destructive methods to prevent the fungi from making aflatoxin by interrupting one of the biochemical steps of aflatoxin biosynthesis. In this report, we identified a gene for an esterase that is involved in a middle step in the biosynthesis process. We show that this enzyme is necessary for the conversion of the aflatoxin precursors versiconal hemiacetal acetate to versiconal and versiconal acetate to versiconol, respectively. Further work may identify cultural practices or resistant corn or cottonseed that could reduce aflatoxin contamination of crops in the field by interfering with the action of the esterase.

Technical Abstract:
The route for the conversion of 1-hydroxyversicolorone to versiconal hemiacetal acetate (VHA) to versiconal (VHOH) in the aflatoxin biosynthesis involves two metabolic grids. A VHA reductase, a monooxygenase, and a VHA esterase are believed to carry out the conversion of related compounds in the grids. We deleted the estA gene in the aflatoxin gene cluster of A. parasiticus SRRC 2043, an O-methylstergimatocystin (OMST)-accumulating isolate. The estA- deleted mutants had a pigmented phenotype and accumulated mainly HVA, a small amount of versiconol acetate (VOAc), and other downstream aflatoxin intermediates, including VHOH, versicolorin A, versicolorin B and OMST. Addition of dichlorvos, an inhibitor of acetyl choline esterase, to an estA-deleted mutant blocked the production of the mentioned downstream intermediates. Cell-free extracts of estA deleted mutants converted VHA to VHOH and VOAc to VOH at efficiencies 10% and 15%, respectively, that of the estA-containing parental strain. These results show that other aflatoxin-pathway nonspecific esterase(s) play a minor role during aflatoxin biosynthesis and that the estA-encoded esterase accounts for at least 85 to 90% of the conversion of VHA to VHOH and VOAc to VOH.